DISCUSSION
Majority of human genetic diseases arise from point mutations.
G:C>A:T or T:A>C:G point mutations represent
nearly half of all pathogenic single-nucleotide polymorphisms (SNPs)
(Gaudelli et al., 2017;
Zhiquan Liu et al., 2018). While most
animal models are generated by traditional gene knockout, which is
time-consuming and costly while CRISPR/Cas9 system gives unpredictable
deletions. Generation of point mutation disease model is most time
consuming with low success rate (Zhiquan
Liu et al., 2018; Zhang et al., 2017).
However, point mutation mouse models are the best human disease models
which can precisely mimic human pathology. Previous reports have
demonstrated that cytosine base editing (CBE) systems are versatile in
different animal models and plants
(Zhiquan Liu et al., 2018;
Zhang et al., 2017;
Zong et al., 2017;
Li et al., 2017;
G. Yang et al., 2018). Moreover, BE is a
secure system with less off-target effects
(Komor, Kim, Packer, Zuris, & Liu, 2016;
D. Kim et al., 2017) and can modify
genomic DNA without double-strand breaks (DSB). Yet, applications of
base substitutions in generation of animal models are still limited.
David Liu has developed a variety of versions of base editing systems
but the newest system BE4 has not been tested in animal models.
BE-mediated STOP-codon disrupts genes by converting C to T in
coding sequences (CAG, CAA, CGA) and leads to a stop codon, providing a
secure approach to generate knockout animal models but with minimum
interference of genome structure. It is similar to many human genetic
diseases (G. Yang et al., 2018;
Kuscu et al., 2017;
Billon et al., 2017). In this study, we
have applied BE4 plasmid along with sgRNA expression plasmid in
transgenic microinjection. We designed a precise base editing method
which knockout tyrosinase gene and results in
loss-of-pigmentation (Albinism). We achieved successful C>T
transition with high efficiency. C>T conversions have
occurred exclusively within the approximate editing window of
protospacers (positions~4–8). Our results highlighted
that BE4 system can introduce site-specific and single-base substitution
with high precision and efficiency in mouse embryos with no off-target
mutation. This adds great values to human disease modeling.